Tremendous advances in genomics during the past two decades hold great potential for the development of new health innovations. For this potential to be realized in the form of advances in biomedical research and novel prevention, diagnosis, treatment and disease management strategies, we must overcome one technological barrier, namely the ability to perform reliable molecular profiling analyses of low quantity cells/nucleic acid material. Current technologies are not suitable for most low-quantity nucleic applications, and require multiple nucleic acid manipulation and amplification steps that are known to introduce biases and artifacts that confound downstream analyses and reduce/eliminate quantitative power. This grant application proposes various strategies for picogram-level DNA and RNA samples to be profiled in an unbiased and amplification-free, high-throughput manner, and paves the way towards single cell measurements. In Phase I, we will set the groundwork for unprecedented technologies enabling attomole-level, amplification-free high- throughput genomics tools. The Phase I specific aims are: (1) Optimization of DNA tailing and sequencing surface capture steps, (2) Development of DNA fragmentation strategies suitable for low DNA quantities, (3) Specific capture and sequencing of polyA+ mRNA from total RNA, and (4) Development of single-step sequence selection and single molecule sequencing technology. In Phase II, we will expand our work from Phase I to develop mature procedures ready for commercialization. The Phase II specific aims are: (1) High- throughput sequencing of limiting DNA samples, (2) Sequencing minute polyA+ RNA quantities using direct RNA sequencing, (3) Optimization of tailing and surface capture steps for direct RNA sequencing, (4) cDNA- based low-quantity RNA sequencing on surface, and (5) Development of a sequencing strategy for damaged and limited quantity nucleic acid samples. These advancements will enable various long desired and needed studies, open new research frontiers and provide a comprehensive understanding of the biological mechanisms underlying disease states, such as cancer, heart disease, diabetes, and others, ultimately leading to revolutionary new ways to diagnose, treat and prevent human disease. PUBLIC HEALTH RELEVANCE: The sequencing of the first human genome was an unprecedented scientific achievement derived from 13 years of effort by an international coalition of scientists and some $3 billion in funds. The availability of a complete human genome sequence has facilitated research by providing a framework for the genome, now being used for further investigation into the biological mechanisms underlying human disease. Technological advancements now enable sequencing of genomes at a fraction of the time and cost, and the widespread application of high-throughput sequencing technologies has transformed the biomedical research field. However, several fundamental technical shortcomings still remain. Among these limitations, arguably the most critical one is the requirement for high-quantities of valuable input material, namely DNA/RNA. Progress in many research areas, such as, but not limited to, stem cell biology, microbiology, cancer, paleoarcheology, forensics, and clinical diagnostics, is severely impeded by our inability to perform comprehensive and reliable molecular profiling analyses on low-quantity cell and nucleic acid samples. If we are to successfully translate this research knowledge of genome biology to better diagnosing and treating human disease, we must reliably use and analyze minute quantities of nucleic acid derived from patient specimens. This grant application proposes various strategies for picogram-level DNA and RNA samples to be profiled in an unbiased and amplification-free, high-throughput manner, and paves the way towards single cell measurements. These advancements will enable various long desired and needed studies, open new research frontiers and provide a comprehensive understanding of the biological mechanisms underlying disease states, such as cancer, heart disease, diabetes, and others, ultimately leading to revolutionary new ways to diagnose, treat and prevent human disease.